Unveiling allelopathic dynamics and impacts of invasive Erigeron bonariensis and Bidens pilosa on plant communities and soil parameters

Invasive alien species are becoming more and more prevalent worldwide, Erigeron bonariensis and Bidens pilosa are two invasive species of Asteraceae in Egypt. To mitigate their detrimental effects and understand their differences in invasiveness, we compared the allelopathic potentials of E. bonariensis and B. pilosa using leachates, decaying residues, and volatilization processes. Notably, the allelopathic variances in leachates were significant, influenced by plant types, concentrations, and response patterns of target plant traits, as indicated by EC50. The relative phytotoxicity of the invasive species decayed residues peaked between 20 and 25 days in the soil, with a positive correlation with concentrations and soil properties. The highest quantities of phenolic acids were chlorogenic acid and caffeic acid reaching (5.41 and 4.39 µg g−1) E. bonariensis and (4.53 and 4.46 µg g−1) B. pilosa, in leachates extracts respectively, while in the soil extract of decayed residues were coumaric acid and ferulic acid measuring (1.66 and 1.67 µg g−1) E. bonariensis and (1.47 and 1.57 µg g−1) B. pilosa, respectively. Using GC/MS analysis, the main volatile components in E. bonariensis were 1, 8 cineole (5.62%), and α-terpinene (5.43%) and iso-Caryophyllene (5.2%) which showed the greatest inhibitory effects. While B. pilosa main constituents were trans-sabinene (5.39%) and Camphene (5.11%), respectively. Finally, the high invasion level displayed from E. bonariensis (0.221) compared with B. pilosa (0.094) which correlated with the stronger allelopathic activities against plant species, and soil properties. Therefore, the allelopathic potentialities of these species are critically relevant to their invasion success.


Plant material
Invasive Erigeron bonariensis (L.) Cronquist and Bidens pilosa (L.) were harvested in the wild before the flowering stage during 2020-2022, from Al-Beheria and Al-Qalibia governorates, Egypt respectively.The identification was confirmed by plant specialist Dr. Emad Abdel-Kader Desert Research Center, Cairo, Egypt.A voucher samples of Erigeron bonariensis (CAIH-16-9-2020-C) and Bidens pilosa (CAIH-11-7-2020-B) has been deposited at the Herbarium of DRC, Cairo, Egypt.The plants were dried in the shade, chopped, ground into a fine powder, and then stored in paper bags at room temperature.Convolvulus arvensis L., Portulaca oleracea L., and Echinochloa crus-galli (L.) P.Beauv.were collected from Zea mays field in Maryut research station, Desert Research Center.These species represent monocots and dicots to detect the response pattern to allelopathic potentials of E. bonariensis and B. pilosa species.

Analysis of E. bonariensis and B. pilosa communities within invaded sites
Primary surveys were conducted about Erigeron bonariensis and Bidens pilosa invasive weeds at 15 and 5 governorates of Egypt in random patterns during 2020 and 2022 to identify the associated community according to Thomas et al. 41 .The plant species were counted across quadrates (100 × 100 cm 2 ) of invaded sites.The data of assemblages were then presented in density (plants m −2 ) for usage as a function of richness and diversity.The invasion level was represented by the invasion intensity index (III) = P i /MaxP i, where P i represents the observed relative abundance of alien species in one surveyed quadrat and MaxP i represents the maximum relative abundance of alien species among all surveyed quadrats, respectively 42 .Richness, Shannon-wiener index, and Simpson index of diversity were measured according to Margalef 43 , while evenness was quantified according to Pielous 44 , and Magrurran 45 to provide more information about individual distributions.

Leachates of E. bonariensis and B. pilosa allelochemicals
The aboveground parts of E. bonariensis and B. pilosa were extracted by soaking 200 g of the ground parts in 1000 ml of distilled water.This mixture was placed on an orbital shaker at 160 RPM for 12 h at laboratory temperature.Then the extract was strained through cheesecloth to remove plant materials, centrifuged at 3000 RPM for 15 min, filtrated and sterilized using a 0.22 µm pore micro-filter before bioassays.To determine the allelopathic compounds, these water extracts were acidulated to a pH value less than 5 and then partitioned with

Quantitative characterization of allelochemicals by LC-DAD/MS analysis
The analysis of phenolic acids involved dissolving ethyl acetate extract and soil phenolic extracts in methanol (HPLC grade) before injection into LC-DAD electrospray ionization (ESI)-MS analysis (Waters, USA) at Ain Shams University, equipped with a DAD detector (Waters Corporation, Milford MA01757, USA).Compounds were separated using a 150 × 4.6 mm C 18 column.UV/Vis spectra were recorded in the 190-600 nm range and the chromatograms were acquired at 220, 240, 280, 330 and 350 nm.The samples were analyzed by gradient elution at a flow rate of 0.2 ml/min.The mobile phase was a multistep linear solvent gradient system, starting from 100% H 2 O (adjusted to pH 3.2 by HCOOH) up to 100% CH 3 CN in 30 min.The profile and content of phenolic compounds of Hydroxybenzoic acid, cinnamic acid, ferulic acid, coumaric acids, chlorogenic acid, caffeic acid, sinapic acid, vanillic acid, protocatechuic acid, syringic acid, catechin, kaempferol, and quercetin were determined according to the method described previously 49,50 .

Volatilization of allelochemicals from invasive E. bonariensis and B. pilosa parts
The shoots of both E. bonariensis and B. pilosa plants were harvested, and the dried canopy samples were extracted by hydrodistillation 51 .The tested Z. mays crop, C. arvensis, P. oleracea, and E. crus-galli weeds were sterilized and treated with concentrations of 0, 5.0, 10.0, 20 0 µl/ml.Petri dishes were sealed with parafilm and kept at 25 ± 2 °C and then after 7 days seed germination and seedling growth (radical and hypocotyl) were measured.Essential oil was subjected to GC-Ms analysis at The National Research Center.Qualitative identification of the oil constituents was carried out by comparing the retention times and mass fragmentation with computer matching of authentic samples and with published data 52 .

Statistics analysis
The allelopathic effects of B. pilosa and E. bonariensis leachates, decaying and volatile compounds on the target plants were compared using the ANOVA test to separate the effect of plant species, concentration and other variations.Where F test indicated significant differences (P > 0.05) and followed by Duncan multiple range using SPSS, 19 software (SPSS, Chicago, IL USA).The experimental design was a Complete Randomized Design with four replications and repeated more than one time.Additional data, including EC and pH, were entered for statistical analysis using ANOVA (SPSS, Chicago, IL, USA).Correlation analyses were conducted to test the association between EC, pH, and target plant parameters, serving as response determiners for allelopathic potentials.

Guidelines of material collections and studies
All the steps of experimentation on three invasive alien species Asteraceae including Conyza bonariensis and Bidens pilosa, wild weeds, including the collection of plant material, are in compliance with relevant Institutional, National, and International guidelines.The studies were conducted in accordance with local legislation and with permissions from our institutes and complied with the IUCN Policy Statement.

E. bonariensis and B. pilosa spreading and associated weeds relative density in invaded localities
According to surveys, E. bonariensis was associated with 16 species of 11 families and achieved an invasion intensity index of 0.221, while B. pilosa was associated with 19 species of 11 families and recorded an invasion intensity index of 0.094, resulting in a similarity coefficient of 81.39% within the invaded community.For E. bonariensis, Echinochloa colonum had the highest relative density, accounting for 11 www.nature.com/scientificreports/

Allelopathic potentials of E. bonariensis and B. pilosa decayed residues in Z. mays and P. oleracea
The aboveground parts of E. bonariensis and B. pilosa were decayed in sandy soil for 50 days under the greenhouse to measure their allelopathic potentials against other plants and soil properties.The phytotoxicity analysis differentiated E. bonariensis and B. pilosa decayed residues, which showed an increase from 5 to 25 days based on the plant species and concentration in vigor index (germination × (shoot length + root length) of Z. mays and P. oleracea (Fig. 1).As for Z. mays response to decayed residues, gradually decreasing was recorded in growth traits with a significant interaction in shoot length (F = 5.05, P ≤ 0.00) plant species × concentration and (F = 4.37, P ≤ 0.00) time × concentration.These interactions were significant in root length (F = 1327.54,P ≤ 0.00) concentration and interaction of (F = 3.541, P ≤ 0.00) plant species × concentration and (F = 2.48, P ≤ 0.02) time × concentration.Also,  www.nature.com/scientificreports/ it was significant in germination (F = 2.576, P ≤ 0.03) time, (F = 244.04,P ≤ 0.00) concentration and interaction (F = 38.215,P ≤ 0.00) plant species × concentration and (F = 6.529,P ≤ 0.00) time × concentration respectively.As for P. oleracea, in response to decayed residues, A gradual decrease was recorded after 10, 15, and 25 days of decayed residues in shoot length, with significant interaction effects observed for plant species × concentration (F = 11.04,P ≤ 0.00) and time × concentration (F = 16.73,P ≤ 0.00).In root length, there was a significant interaction effect for plant species × concentration (F = 46.79,P ≤ 0.00) and time × concentration (F = 2.659, P ≤ 0.01).Additionally, in germination, significant interaction effects were observed for plant species × concentration (F = 4.06, P ≤ 0.00) and time × concentration (F = 56.815,P ≤ 0.00).
The decayed materials of E. bonariensis and B. pilosa showed a significant effect on Z. mays soil pH (7.60, P ≤ 0.00) time, (10.49, P ≤ 0.00), concentration, and interaction (F = 6.82,P ≤ 0.00) time × concentration.The decayed materials induced a minor gradient increase from the control to the highest concentration in pH value of P. oleracea soil pH (F = 3.80, P ≤ 0.00) time, (16.51, P ≤ 0.00), concentration, and interaction (F = 3.28, P ≤ 0.00) time × concentration respectively (Fig. 2).The decayed materials of E. bonariensis and B. pilosa in sandy soil cultivated with Z. mays and P. oleracea had increased soil EC compared to the control, and this increase was proportional to the residue concentrations.There was a significant effect on Z. mays soil EC (F = 3.21, P ≤ 0.00) in terms of time, (F = 13.25,P ≤ 0.00), and concentration, respectively.The decayed materials also showed significant effects on soil EC cultivated with P. oleracea concerning from time (F = 4.188, P ≤ 0.00) and concentration (12.683,P ≤ 0.00), with an increase from the control to the highest concentration (Fig. 3).

Qualitative and quantitative determination of E. bonariensis and B. pilosa allelochemicals via leachates and decayed residues
Fourteen phenolic compounds were quantified in the leachates and soil incorporated with decayed materials of the two invasive species using LC/MS (Table 4).Initially, aqueous leachates were extracted by ethyl acetate, dried, and dissolved in methanol for chromatographic analysis.The results revealed that the highest amounts of phenolic acids were chlorogenic acid and caffeic acid, reaching (5.41 and 4.39 µg g −1 ) and (4.53 and 4.46 µg g −1 ) in E. bonariensis and B. pilosa extracts, respectively.Moderate amounts were observed for hydroxybenzoic, vanillic, gallic, ferulic, protocatechuic, coumaric, sinapic, kaempferol, and cinnamic acids, while protocatechuic acid and catechin were present in low quantities (1.47 and 1.54 μg g −1 ) and (1.27 and 1.91 μg g −1 ) in E. bonariensis and B. pilosa extracts, respectively.
Regarding decayed residues in soil samples, the phenolic acids analysis, revealed that coumaric acid and ferulic acid were the predominant compounds with concentrations of (1.66 and 1.67 µg g −1 ) and (1.47 and 1.57 µg g −1 ) in E. bonariensis and B. pilosa decayed materials, respectively.Additionally, quercetin was detected in relatively low quantities, measuring 0.18 and 0.19% μg g −1 in E. bonariensis and B. pilosa decayed materials, respectively.

Allelopathic potentials of E. bonariensis and B. pilosa via volatile oils in Z. mays and associated weeds
The allelopathic capabilities of E. bonariensis and B. pilosa volatile oils compounds obtained from hydrodistillation were evaluated on Z. mays, C. arvensis, P. oleracea, and E. crus-galli.Based on EC 50 values, these volatile oils were effective at low concentrations, particularly in the tested species.As for E. bonariensis volatile oil, the Vigor index  www.nature.com/scientificreports/

Discussion
E. bonariensis and B. pilosa are two invasive Asteraceae species that differed in the invasion and impacts in cultivated lands of Egypt.Therefore, to understand their detrimental effects we compare their allelopathic effects through leachate by water extraction, decayed residues in soil and volatilization via their essential oils.Also their effects on the invaded ecosystem were evaluated via the ecological index of richness, diversity and evenness.In the studied invaded communities, E. bonariensis has lower richness and diversity and evenness as compared with B. pilosa.E. bonariensis was found in 15 governorates in croplands, orchards, and wastelands, while B. pilosa was found in croplands across 5 governorates of Egypt.In contrast, B. pilosa was recorded only in cropland www.nature.com/scientificreports/habitats 53 .Invasive alien species are a major threat to global biodiversity loss because of their ability to adapt and flourish in diverse environments 54 .Additionally, the allelopathic impacts of these invasive species were evaluated against different species and soil properties, and their allelochemicals were quantified.Invasive weeds may exert a negative impact on other plant species and soil processes driven by allelopathy or nutrient availability 55 and influence soil physical and chemical properties 56 and nutrient cycle in the ecosystem 57 .Additionally, Allelopathic substances are proposed as an environment-friendly option to lessen the deterioration of ecosystem services 58 .

The invasive E. bonariensis and B. pilosa species allelopathic potentials via leachates against varied species
Generally, Based on EC 50 , E. bonariensis has greater allelopathic activity than B. pilosa through aqueous and organic extracts with varied response patterns in the tested species proportional to the concentrations.According to statistical analysis, there were significant differences in plant species, concentrations and trait response, particularly in seed germination and seedling growth.All plants seem to be leachable (the removal of substances from plants by the aqueous solutes action in different degrees 59 ).Phytotoxicity can be attributed to the characteristics of the material 60 .According to ED 50, The most sensitive among the four tested plants was P. oleracea.However, Z. mays was less sensitive to both two extracts.In this respect, the response indices of root length were found to be more susceptible than other measured parameters to the liberated allelochemicals from both extracts.The dose of allelopathic potentials provides valuable biological insights into the invasive impacts of species 61 .
Root growth is a more sensitive indicator of phytotoxicity than hypocotyl length 62 .The direct contact between the root and phytotoxic compounds present in the extract might inhibit cell division in the growing root tip 63 .
Allelopathy can be an important component of crop/weed interference 64 .Crop plants were more strongly affected by invasive species extracts than weeds 35 .

The invasive E. bonariensis and B. pilosa species allelopathic impacts during the decaying process against plant species and soil properties
The invasive species vegetative parts were decayed in the soil to test the biological activity and assess their impacts on the soil parameters.The decayed process displayed a periodic increase over time starting from 5 days and reaching its peaks at 20 and 25 days.Subsequently, a decline in phytotoxic effects was observed from 30 days onwards, diminishing by the end of the decay period (50 days).There were dramatic significant patterns in plant species, concentrations and times.The response pattern of P. oleracea was higher than Z. mays to decayed allelochemicals.There were slight differences between E. bonariensis and B. pilosa allelopathic abilities on the tested plant and the soil parameters.Simultaneously, a positive correlation was identified between soil physicochemical properties and the response of plant growth parameters.As for Z. mays trails, the vigor index showed a correlation of 0.595 with decayed residue concentration for E. bonariensis and 0.538 for B. pilosa.Additionally, Z. mays soil pH exhibited a correlation of 0.758 with decayed residue concentration for E. bonariensis and 0.791 for B. pilosa.The correlation between Z. mays soil EC values and decayed residue concentration was 0.759 for E. bonariensis and 0.819 for B. pilosa, respectively.As for P. oleracea trails, there are positive correlations with the plant vigor index and decayed residue between concentrations (0.433) E. bonariensis and (0.055) B. pilosa respectively.Similarly, decayed residues exhibited correlations between soil pH and concentration of 0.718 (E.bonariensis) and 0.626 (B.pilosa) respectively.Additionally, the correlation between soil EC values and decayed residue concentration was 0.595 (E.bonariensis) and 0.339 (B.pilosa), respectively.Soil plays a crucial role as a biological environment with the potential to detoxify or toxify allelochemicals through microbial action 65 .The decaying weed residues effects depend upon the release of allelochemicals from them into the soil causing adverse effects on other plants 66,67 .The deleterious effect of decaying weed residues on the growth and yield of subsequent crops in the field was reported 68 .High nutrient availability often observed in plant invasions may be driven in part by the rapid decomposition of exotic plant litter 69 .It is important to identify the allelopathic compounds in soil or water substrates 70 .The most effective allelochemicals have very limited water solubility 71 .Soil incorporation with crop residues resulted in an overall decline in the density and vigor of the weed community 72 .Residue-mediated inhibition can occur only if the susceptibility period of the receptor plant coincides with the inhibitory allelopathic potential peak period 73 .Timing of phytotoxicity is variable, with some reporting it is greatest at early 74,75 or increasing toxicity with increasing time after incorporation 76 .The changes over time in both the composition and quantity of allelochemicals can either increase or decrease the phytotoxicity 77 .

E. bonariensis and B. pilosa species allelopathic impacts via volatilization against different species
Here, we test the allelopathic ability of volatile compounds liberated from the studied invasive weeds.The highest amounts of inhibition based on EC 50 revealed that E. bonariensis essential oils had supreme inhibitory effects over B. pilosa against the tested plants.While, the incidence of growth inhibition was distinguished in the plant species and used concentration as well as the plant traits, and root length was more sensitive than other parameters.Therefore, the allelopathic potential of essential oils demonstrates high inhibition properties towards the selected weeds, compared with the response of the tested crop.These results highlight the influence of invasive plant species volatile oils in the invaded areas.However, these results presented an added value of invasive species essential oil that exhibited weed suppression and can be used as an alternative means to synthetic herbicides.Chemically, invasive plants can modify their environment by releasing secondary metabolites, such as root exudates (liquid) or (gaseous) volatile organic compounds 78 .Allelopathy can regulate plant biodiversity through its impact on plant adaptation, survival, and community organization 79 .However, the effect of allelopathy is not solely harmful; beneficial aspects, such as weed control, are also possible 80 .
Vol www.nature.com/scientificreports/ The ways of allelochemicals in the environment and the effects they have on soil and plant communities Both E. bonariensis and B. pilosa invasive species demonstrated potent allelopathic efficacy via leachates, decaying, and essential oils on different species, while these allelopathic potentials of aqueous leachates and decayed residues related to phenolic compounds.The Asteraceae family is considered a repository of species to be explored for allelopathy with several associated secondary metabolites such as terpenes, saponins, alkaloids, alkamides, cinnamic acid derivatives, and flavonoids 81 .Phenolic acids are a diverse class of compounds that can act as agents in plant defense 82 .Exotic plants can successfully establish communities due to their relatively strong allelopathic effects in the invaded habitats 83 .Allelopathy of knotweeds may contribute to establishing their new habitats in the introduced ranges as invasive plant species 84 .
The suppressive potential of leachates, decayed residues, and volatilization is influenced by the species, concentration, and response traits of the target species.Stronger detrimental impacts were seen from essential oil followed by leachates compared to decayed residues.Nevertheless, decayed residues displayed a significant negative impact on soil properties, specifically on EC and pH values.However, there are positive correlations between the response of plant parameters and the decayed soil physicochemical properties of EC and pH values.Allelopathy and allelochemicals have provided fascinating insights into plant-plant interactions and their consequences for biodiversity, productivity and sustainability 85 , and could be utilized in conventional or organic agriculture 86 .On the other hand, the high invasion level of E. bonariensis joint with allelopathic effectiveness and by low richness and Simpson index 1 (λ) and vice versa in B. pilosa species.This allelopathic potentiality proved the strong invasive nature of E. bonariensis and impacts on the native plant biodiversity compared to B. pilosa species.

Conclusion
The characteristics of E. bonariensis and B. pilosa invasive species are revealed by leachates, decomposing residues, and volatile compounds, which are employed as distinct threats to the native species and agricultural soil.The key allelochemicals known to be involved are phenolic compounds via leachates and decayed residues Furthermore, volatile substances were more suppressive than leachates followed by decayed residues.Conversely, E. bonariensis, showed highly invasive species and more allelopathic activity than B. pilosa species, affecting a wider range of plant species and soil characteristics.Therefore, understanding these allelopathic potentials is crucial for preventing the invasion and impacts on ecosystems and crop productivity and implementing strategic management of invasive species.

Table 1 ). Allelopathic potentials of E. bonariensis and B. pilosa leachates via water extracts
.16% across croplands, orchards, and wastelands in 15 governorates.In the invaded B. pilosa community, Bromus catharticus had the highest relative density, representing 11.34% in croplands across 5 governorates.The richness R1 and R2 parameters was higher

Table 1 .
Richness, similarity, diversity, and evenness of E. bonariensis and B. pilosa in invaded localities.

Table 2 .
Allelopathic abilities of E. bonariensis and B.pilosa water extracts based on EC 50 (g dry wt.100 ml −1 water) on different species.

Table 4 .
Phenolic acids liberated from leachates in water extracted by ethyl acetate (µg g −1 crude extract) and decayed in soil (µg 200 g soil) analyzed by LC-DAD/MS.

Table 5 .
Allelopathic abilities of E. bonariensis and B. pilosa volatile essential oils based on EC 50 (µl ml −1 ) on maize and some associated weeds germination and seedling development.

Table 6 .
Composition and percentages of E. bonariensis and B. pilosa (Shoot parts) essential oils analyzed by GC/MS.